A quantitative detection kit for allergen-specific IgE antibodies based on liquid phase chip method and its application

Abstract

The present invention belongs to the field of biological detection, and discloses an allergen-specific IgE antibody quantitative detection kit based on a liquid phase chip method and its application. The kit is based on a liquid phase chip method, and the carrier particles used are assembled microspheres, which can significantly improve the sensitivity of detection and reduce nonspecific adsorption; the present invention coats common allergens and their allergen component antigens on different coded microspheres respectively, and realizes single-tube detection of multiple indicators. The present invention also discloses the application of the above antibody detection reagent. It has the advantages of small sample usage, high sensitivity, high throughput, high degree of automation, etc.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an allergen-specific IgE antibody quantitative detection kit based on a liquid phase chip method and an application thereof. Background Art

[0002] The allergen-specific IgE antibody detection kit is used to detect allergen-specific IgE antibodies. Detection of allergen-specific IgE antibodies can be used as the most commonly used auxiliary diagnostic method in type I allergy.

[0003] Allergy diagnosis includes clinical history, skin test and allergen-specific IgE test. Only when the three are consistent can the diagnosis be confirmed. A positive result of specific IgE can help identify specific allergens, take further avoidance measures, and provide a warning about the risk of clinical reaction to allergens. The clinical value of a negative result is that it can rule out the symptoms and causes of allergies and provide appropriate treatment plans based on objective facts.

[0004] According to the difference in solid support and reporter molecule, the methods for detecting allergen-specific IgE antibodies can be divided into three categories: (1) fluorescent enzyme-linked immunosorbent assay, developed by Phadia of Sweden, ImmunoCAP, which uses cellulose derivatives as solid support, has the advantages of a wide variety of allergens, accurate results, high clinical recognition, quantification, and high degree of automation, but is relatively expensive; (2) enzyme-linked immunosorbent assay or chemiluminescence method, usually using micropores or microspheres as solid support, and alkaline phosphatase or acridinium ester as reporter molecule, which is also one of the most common methods; (3) immunoblotting and protein chip method are to adsorb allergens onto membranes or glass, and distinguish different types of allergens by the different positions of allergens on the solid support. Methods (1) and (2) can detect allergen-specific IgE antibodies quantitatively or semi-quantitatively, but their main technical disadvantages are that each reaction can only detect one indicator and use a large amount of serum sample. Although method (3) can theoretically detect multiple indicators at one time, the immunochemical reaction occurs between the solid phase and the liquid phase, has poor repeatability, low sensitivity, and unstable results. It can only perform qualitative detection and uses a large amount of sample.

[0005] Liquid chip method (also called flow fluorescence luminescence method) for allergen detection has been developed in recent years. Compared with traditional detection methods, it has the advantages of high-throughput quantitative testing, high sensitivity, wide linear range, fast reaction, automation, good repeatability, etc. Its core lies in the liquid chip system, of which the coded microspheres are its key technology. The principle is to act as a carrier for the reaction of biological molecules such as proteins and nucleic acids. The coded microsphere technology was originally developed by Luminex, an American company. It uses a variety of fluorescent substances to form a series of coded microspheres as carriers of related biological molecular reactions. The reaction is carried out in a liquid environment that is more conducive to maintaining the natural conformation of proteins, and can achieve high-throughput and multiple detection of biological molecules such as proteins and nucleic acids. Although the early detection method has the advantages of using a small amount of sample and being able to detect multiple indicators at the same time, it has the following disadvantages: the sensitivity is not as good as the fluorescent enzyme-linked immunosorbent assay or chemiluminescence method; it can achieve secondary diagnosis (represented by extract allergens), but has not achieved tertiary diagnosis (represented by single molecules). Summary of the invention

[0006] In view of this, the present invention first proposes an allergen-specific IgE antibody quantitative detection kit based on the liquid chip method. The kit is based on the liquid chip method, and the carrier particles used adopt host-guest structure assembled microspheres and multi-fluorescence channel encoding, which can significantly improve the sensitivity of detection and reduce nonspecific adsorption; the present invention coats common allergens and their allergen component antigens on different coded microspheres respectively, and multiple indicators are detected in the same tube, with small sample usage, high throughput and high degree of automation, providing a comprehensive, accurate and convenient clinical detection method for the diagnosis of allergic diseases.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions to achieve the above problems:

[0008] An allergen-specific IgE antibody quantitative detection kit based on a liquid phase chip method, comprising a mixture of coded microspheres coated with different capture probes, a reaction buffer, a biotin marker, a fluorescent conjugate, a calibrator and a quality control product;

[0009] In some specific embodiments of the present invention, the coded microsphere mixture is a mixture of antigen-coated coded microspheres and quality control-coated coded microspheres, or a mixture of anti-human IgE monoclonal antibody-coated coded microspheres and quality control-coated coded microspheres;

[0010] In some specific embodiments of the present invention, the coded microspheres provide a carrier particle based on quantum dot coding and assembly microspheres, which includes a mother ball and a daughter ball located on the surface of the mother ball; the mother ball and / or at least one daughter ball includes at least one type of quantum dots to achieve multiple fluorescent coding. The mother ball and / or the daughter ball include magnetic nanoparticles, so that both the mother ball and the daughter ball have magnetism, or one of the mother ball and the daughter ball has magnetism.

[0011] In some specific embodiments of the present invention, the capture probes coated on the surface of the coded microspheres in the coded microsphere mixture are antigens; the antigen-coated coded microspheres are coded microspheres whose capture probes are coated with an allergen or an allergen component antigen, and different types of capture probes correspond to different fluorescent coded microspheres.

[0012] Preferably, the allergens are generally derived from the following substances or their extracts: house dust mites, dust mites, cockroaches, Aspergillus fumigatus, Alternaria alternata, cat dander, dog dander, common ragweed, mugwort, timothy grass, Bermuda grass, birch, maple sycamore, willow, egg white, egg yolk, milk, cod, wheat, crab, shrimp, beef, lamb and cross-reactive carbohydrate determinants.

[0013] Preferably, the allergen component antigens are highly purified natural extracted antigens or genetically engineered recombinant antigens. The following component antigens are commonly used (using WHO allergen nomenclature): Derp 1, Der p 2, Der p 10, Der p23, Fel d 1, Can f 1, Art v 1, Gal d 1, Gal d 2, Gal d 3, Gal d4, Bos d4, Bos d 5, Bosd 6, Bos d 8, etc.

[0014] Preferably, the capture probe coated on the surface of the anti-human IgE monoclonal antibody-coated encoded microspheres is an anti-human IgE monoclonal antibody, and the anti-human IgE monoclonal antibody-coated encoded microspheres are used for specific IgE calibration curves.

[0015] Preferably, the allergen is an allergen modified by a hydrophilic polymer, and the two are combined to form a hydrophilic polymer-allergen complex, the hydrophilic polymer is selected from poly-L-lysine, nona-arginine, polyethyleneimine, preferably poly-L-lysine, and its molecular weight is 1000-20000Da; the molar ratio of the hydrophilic polymer to the allergen extract is 1:50-1:300;

[0016] The method for preparing the allergen-coated coded microsphere mixture comprises the following steps:

[0017] Add the encoded microspheres (such as tosyl encoded microspheres) and coating buffer (0.1 mol / L Borate buffer pH 9.5) to the microtube, and vortex mix them with a mixer, remove the supernatant after magnetic separation; then add 0.1 mol / L coating buffer and mix them, add allergens or allergen component antigens and mix them; add reaction catalytic solution (3 mol / LAmmonium sulfate / 0.1 mol / LBorate buffer pH 9.5), and mix them at 37°C for 18 hours;

[0018] Add blocking agent (1% human serum albumin and 5% glycine) and mix at 37°C for more than 6 hours; remove the supernatant after magnetic separation, add washing buffer (phosphate buffer containing Tween20), and repeat washing 3 times; resuspend with coded microsphere diluent and store at 2-8°C.

[0019] Preferably, the coded microspheres also have a quality control coded microsphere system, which includes multiple quality control coded microspheres. The quality control coded microsphere system and the detection coded microsphere system (a mixture of coded microspheres coated with different capture probes) are in the same reaction tube and are independent of each other. The quality control coded microspheres include internal quality control coded microspheres and blank control quality control coded microspheres. The internal quality control coded microspheres are used for positive quality control, and the blank control quality control coded microspheres are used for negative quality control.

[0020] In some specific embodiments of the present invention, the liquid chip detection reagents can be flexibly combined and simplified according to specific circumstances when used. For example, if only some allergen-specific antibodies are detected, only one allergen probe or a selective combination of multiple allergen probes can be used.

[0021] In some specific embodiments of the present invention, the fluorescent conjugate is allophycocyanin (APC) or Alexa Fluor 647 or Cy5-labeled streptavidin;

[0022] The fluorescent conjugate is preferably streptavidin labeled with allophycocyanin, and its preparation method comprises: mixing the following components in percentage by weight: 0.5-3% human serum albumin, 1-10% trehalose, 0.05-0.2% Tween-20, 0.06-0.3% Proclin 300, and then using a certain proportion of deionized water and 0.1M phosphate buffer (pH=7-8) to prepare each index to 0.1-3.0 μg / mL, thus obtaining streptavidin labeled with allophycocyanin;

[0023] Preferably, the biotin marker includes biotin-labeled anti-human IgE antibody and anti-DNP antibody. The biotin-labeled anti-human IgE antibody is a biotin-labeled mouse anti-human IgE antibody, and the concentration of each index of the biotin-labeled anti-human IgE antibody is 0.1-3.0 μg / mL; the biotin-labeled anti-DNP antibody is a biotin-labeled mouse anti-DNP monoclonal antibody, and the concentration of the biotin-labeled anti-DNP antibody is 0.1-3.0 μg / mL;

[0024] Preferably, the reaction buffer contains a phosphate buffer containing Tween-20 and Procline 300;

[0025] Preferably, the calibrator is IgE protein, and the concentrations of the calibrator include 0AU / mL, 0.1AU / mL, 0.35AU / mL, 0.70AU / mL, 3.5AU / mL, 17.5AU / mL, 50AU / mL, and 100AU / mL.

[0026] Preferably, the quality control products are specific IgE curve quality control products and specific IgE quality control products. The specific IgE curve quality control products are IgE proteins. The specific IgE quality control products achieve indoor quality control of allergen-specific IgE through three IgE antibody sera containing high, medium and low concentrations, such as specific IgE multi-negative, house dust mite (D1) IgE antibodies, birch (T3) IgE antibodies, and milk (F2) IgE antibodies.

[0027] In some specific embodiments of the present invention, the detection steps of the allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method of the present invention are:

[0028] S1. Add the sample to be tested (or calibrator and quality control product), reaction buffer, and a mixture of coded microspheres coated with different capture probes to the reaction tube, stir and mix evenly, and react at 37°C;

[0029] S2. After the reaction is completed, magnetic separation is performed, the supernatant is discarded, and a washing buffer is added. After stirring and mixing, magnetic separation is performed again, and the supernatant is discarded again; the washing step is repeated three times in total;

[0030] S3, then add biotin marker to the reaction tube, stir and mix well, and then react at 37°C;

[0031] S4, repeat the cleaning steps of S2 three times in total;

[0032] S5, then add the fluorescent conjugate to the reaction tube, stir and mix well, and then react at 37°C;

[0033] S6, repeat the cleaning step of S2 three times in total;

[0034] S7, add an equal volume of detection solution (phosphate buffer, Proclin300, etc.) to the reaction tube, stir to mix, and place the reaction tube in a flow cytometry luminometer for reading to measure the fluorescence value;

[0035] S8. Using the calibrator to obtain a calibration curve, the iMatrix analysis software uses the fluorescence value into a formula to calculate the concentration of allergen-specific IgE antibodies in the sample to be tested;

[0036] S9, S1~S8 are used in conjunction with the iMatrix fully automatic liquid chip analysis instrument to achieve automated testing.

[0037] The present invention also provides an application of the above-mentioned allergen-specific IgE antibody detection reagent. The reagent is used for quantitative detection of at least one allergen-specific IgE antibody of house dust mite, dust mite, cockroach, Aspergillus fumigatus, Alternaria alternata, cat dander, dog dander, common ragweed, mugwort, timothy grass, Bermuda grass, birch, maple-leaf sycamore, willow, egg white, egg yolk, milk, cod, wheat, crab, shrimp, beef, mutton, cross-reactive carbohydrate determinant, Derp 1, Der p 2, Der p 10, Der p 23, Fel d 1, Can f 1, Art v 1, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Bos d 4, Bos d 5, Bos d 6, Bos d 8.

[0038] The present invention uses assembled microspheres with a host-guest structure formed by connecting nano-sized microspheres to the surface of micron or submicron-sized coded microspheres as solid phase carriers for biological detection, which can significantly improve the sensitivity of detection compared with single smooth-surfaced microspheres. By modifying allergens with hydrophilic polymers to form polymerized allergens, the coating amount of allergens on the surface of magnetically encoded microspheres is increased. By using red fluorescent reporter molecules (such as APC), the sample background is relatively low. Through the above design, the nonspecific background is reduced and the sensitivity is significantly improved.

[0039] The liquid chip detection reagent includes secondary diagnosis (represented by extract allergens) and tertiary diagnosis (represented by single molecules), which can be used clinically for accurate diagnosis and treatment of allergic diseases. It helps to identify the main allergenic components, identify cross-sensitization, and indicate possible cross-reactivity; it helps to indicate components related to severe allergic reactions, improve diagnostic accuracy, and is used to indicate the risk of potential severe reactions, systemic reactions, and mild reactions; it helps to identify specific protein allergenic components to screen specific patients suitable for desensitization immunotherapy and improve the success rate of desensitization immunotherapy; it helps to identify the stability characteristics of component proteins, avoid simple and crude avoidance of food allergens, and provide accurate guidance on healthy and safe diets for patients with food allergies.

[0040] The beneficial effects of the present invention include but are not limited to:

[0041] 1) The kit of the present invention uses magnetically encoded microspheres as solid phase carriers, and adopts assembled microspheres with a topological structure formed by connecting nano-sized microspheres (daughter balls) on the surface of micron or submicron-sized microspheres (mother balls). Compared with single smooth-surfaced microspheres, the assembled microspheres have a larger specific surface area, which can significantly improve the reaction kinetics of identifying and capturing the IgE antibodies to be detected and the ability to capture IgE antibodies, thereby improving the detection sensitivity of the reagent.

[0042] 2) The kit of the present invention utilizes the multifunctional base sites of the hydrophilic polymer to obtain allergens modified with the hydrophilic polymer, further increasing the coating amount of allergens on the surface of the magnetically encoded microspheres, increasing the potential number of allergen-IgE antibody complexes, and improving the sensitivity of the detection system.

[0043] 3) The detection targets of the kit of the present invention include extract allergens (secondary diagnosis) and component antigens (tertiary diagnosis), which can be clinically applied to the precise diagnosis and treatment of allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0045] Figure 1 for Figure 1 This is a clustering diagram of magnetically encoded microspheres detected in Example 10. DETAILED DESCRIPTION

[0046] In order to make the technical personnel in this field understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0047] The allergen-specific IgE antibody kit of the present invention comprises a coded microsphere mixture, a reaction buffer, a biotin marker, a fluorescent conjugate, a calibrator and a quality control product;

[0048] The coded microsphere mixture is a mixture of allergen extracts or allergen component antigen-coated coded microspheres and quality control microspheres, or a mixture of anti-human IgE monoclonal antibody-coated coded microspheres and quality control microspheres.

[0049] 1. Preparation of coded microspheres

[0050] The coded microspheres include a carrier particle based on quantum dot coding and assembly microspheres, which includes a mother ball and a daughter ball located on the surface of the mother ball; the mother ball and / or at least one daughter ball include at least one type of quantum dots. The mother ball and / or the daughter ball include magnetic nanoparticles, that is, both the mother ball and the daughter ball are magnetic, or one of the mother ball and the daughter ball is magnetic.

[0051] The preparation of the coded microspheres of the present invention is mainly composed of two structural units, a mother ball and a daughter ball. The mother ball and the daughter ball are respectively assembled through chemical covalent reactions between the functional groups on their respective surfaces to form assembled microspheres having a raspberry-like structure, such as Figure 1 As shown, the preparation methods of the mother ball and the daughter ball structural unit are described as follows, using quantum dots with two central emission wavelengths, but the following method and encoding concept can also be used, using two or more quantum dots with a central emission wavelength absolute value difference of not less than 30nm, or loading one type of quantum dots inside or on the surface of the mother ball, and loading an organic fluorescent dye inside the daughter ball with a fluorescence central emission wavelength difference of not less than 50nm from the quantum dots loaded on the mother ball. Magnetic polymer microspheres of 5 to 6 μm are preferred, with good suspension properties to achieve automated detection. See patent CN 105219373 B for details.

[0052] 2. Coating Unit and Preparation Method

[0053] Type 1

[0054] The surface of the coded microsphere is coated with a capture probe, and the capture probe coated on the surface is an antigen. The antigen-coated coded microsphere mixture is a mixture of coded microspheres coated with allergens or allergen component antigens.

[0055] The allergens are generally extracted from: house dust mites, dust mites, cockroaches, Aspergillus fumigatus, Alternaria alternata, cat dander, dog dander, common ragweed, mugwort, timothy grass, Bermuda grass, birch, maple sycamore, willow, egg white, egg yolk, milk, cod, wheat, crab, shrimp, beef, lamb and cross-reactive carbohydrate determinants, etc.

[0056] The allergen component antigens are highly purified natural extracted antigens or genetically engineered recombinant antigens. The following component antigens are generally used: Der p 1, Derp 2, Derp 10, Derp 23, Fel d 1, Can f 1, Art v 1, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Bos d 4, Bos d 5, Bos d 6, Bos d 8, etc.

[0057] The allergen is also combined with a hydrophilic polymer to form a hydrophilic polymer-allergen complex, wherein the hydrophilic polymer is selected from poly-L-lysine, nona-arginine, and polyethyleneimine, preferably poly-L-lysine, and has a molecular weight of 1000 to 20000 Da; the molar ratio of the hydrophilic polymer to the allergen extract is 1:50 to 1:300, preferably 1:180 to 1:210;

[0058] Preparation method of detection reagent:

[0059] Step 1: Preparation of the hydrophilic polymer-allergen complex: Take an appropriate amount of allergen and dissolve it in 0.05M~0.2M MES (pH=5.0) buffer, add an excess of freshly prepared EDC solution and an appropriate amount of hydrophilic polymer (poly-L-lysine), vortex mix, place on a constant temperature mixer, incubate at room temperature at 1000 rpm for 0.5~3h, elute with a desalting column after the reaction is completed, and take the eluate. The concentration of the complex is 2mg / mL.

[0060] Step 2: Add the encoded microspheres (such as 6um tosyl encoded microspheres) and 0.1mol / L coating buffer to the microtube, and vortex mix them using a mixer, remove the supernatant after magnetic separation; then add 0.1mol / L coating buffer and mix them, add the hydrophilic polymer-allergen complex or allergen component antigen and mix them; add 3mol / L reaction catalytic solution and mix them at 37°C in the dark for 18 hours;

[0061] Step 3: Preferably, add a blocking agent and mix at 37°C in the dark for more than 6 hours; remove the supernatant after magnetic separation, add washing buffer, and repeat washing 3 times; resuspend with encoded microsphere preservation solution and store at 2-8°C.

[0062] Type 2

[0063] The capture probe coated on the surface of the encoded microsphere is an anti-human IgE monoclonal antibody. The encoded microsphere is used for specific IgE calibration curve. The encoded microsphere of this type is a carboxyl encoded microsphere or a tosyl encoded microsphere, preferably a carboxyl encoded microsphere.

[0064] In some embodiments, a quality control coded microsphere system is also included, including a plurality of quality control microspheres coated with quality control products, wherein the quality control microspheres include at least one of internal quality control microspheres and blank control quality control microspheres. The internal quality control microspheres are used to detect the instrument detection system and reagent conditions, and the blank control quality control microspheres are used to detect the non-specific binding of the sample. The coded microspheres are carboxyl coded microspheres and tosyl coded microspheres, preferably carboxyl coded microspheres.

[0065] Preparation method of anti-human IgE monoclonal antibody coated coded microspheres and quality control microspheres:

[0066] Step 1: Add the encoded microspheres (such as 6um carboxyl encoded microspheres) and 0.05mol / L coating buffer to the microtube, and use a mixer to vortex mix, remove the supernatant after magnetic separation; then add 0.05mol / L coating buffer and mix; add appropriate amounts of EDC and NHS, and activate at 1000rpm at room temperature for 30 minutes; remove the supernatant after magnetic separation, add washing buffer, and repeat washing 3 times; add mouse anti-human IgE monoclonal antibody or DNP-BSA, and mix, mix at room temperature in the dark for 2 hours; repeat washing 3 times.

[0067] Step 2: Add blocking agent and mix well at room temperature in the dark for more than 2 hours; remove the supernatant after magnetic separation, add washing buffer, and repeat washing 3 times; resuspend with encoded microsphere preservation solution and store at 2-8°C.

[0068] The liquid chip detection reagents can be flexibly combined and simplified according to specific circumstances when used. For example, if only some allergen-specific antibodies are detected, only one allergen probe or any combination of multiple allergen probes can be used.

[0069] Example 1

[0070] In the allergen-specific IgE antibody detection kit of this embodiment, the coded microsphere mixture includes a first group of inhaled antigens, which include house dust mite D1, dust mite D2, cockroach I6, Aspergillus fumigatus M3, Alternaria alternata M6, cat dander E1, dog dander E5, common ragweed W1, mugwort W6, Bermuda grass G2, Timothy grass G6, birch T3, maple sycamore T11, willow T12 and cross-reactive carbohydrate determinant CCD antigens. In some specific embodiments, the inhaled group 1 antigens include house dust mite D1, dust mite D2, cockroach I6, Aspergillus fumigatus M3, cat dander E1, dog dander E5, common ragweed W1, mugwort W6 and cross-reactive carbohydrate determinant CCD.

[0071] Example 2

[0072] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a second group of inhaled antigens, and the second group of inhaled antigens includes at least four antigens of Der p 1, Der p 2, Der p 10, Der p 23, Fel d 1, Can f 1, and Art v 1. In some specific embodiments, the inhaled group 2 antigens include Der p 1, Derp2, Derp 10, and Derp 23.

[0073] Example 3

[0074] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a third group of inhaled antigens, and the third group of inhaled antigens includes 6 antigens, namely, house dust mite D1, dust mite D2, Derp 1, Derp 2, Der p 10, and Der p23.

[0075] Example 4

[0076] The coded microsphere mixture of the allergen-specific IgE antibody detection kit includes a fourth group of inhaled antigens, and the fourth group of inhaled antigens includes a first group of inhaled antigens and a second group of inhaled antigens.

[0077] Example 5

[0078] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a first group of food antigens, which includes at least 8 antigens of egg white F1, egg yolk F75, milk F2, cod F3, wheat F4, crab F23, shrimp F24, beef F27, mutton F28 and cross-reactive carbohydrate determinant CCD antigens. In some specific embodiments, the first group of food antigens includes egg white F1, egg yolk F75, milk F2, cod F3, peanut F13, soybean F14, crab F23, shrimp F24 and cross-reactive carbohydrate determinant CCD.

[0079] Example 6

[0080] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a second group of food antigens, and the second group of food antigens includes at least 8 antigens of Gal d 1, Gal d 2, Gal d 3, Gal d 4, Bos d 4, Bos d 5, Bos d 6, and Bos d 8. In some specific embodiments, the food group 1 antigens include Gal d 1, Gal d 2, Gal d 3, Gal d 4, Bos d 4, Bos d 5, Bos d 6, and Bos d 8.

[0081] Example 7

[0082] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a third group of food antigens, and the third group of food antigens includes 5 antigens, namely milk F2, Bos d4, Bos d 5, Bos d 6, and Bos d 8.

[0083] Example 8

[0084] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a fourth group of food antigens, and the fourth group of food antigens includes the first group of food antigens and the second group of food antigens.

[0085] Example 9

[0086] In the detection reagent of the allergen-specific IgE antibody detection kit, the coded microsphere mixture includes a first group of comprehensive antigens, which include a first group of inhaled antigens and a first group of food antigens, specifically including house dust mite D1, dust mite D2, cockroach I6, Aspergillus fumigatus M3, Alternaria alternata M6, cat dander E1, dog dander E5, common ragweed W1, mugwort W6, Bermuda grass G2, Timothy grass G6, birch T3, maple sycamore T11, willow T12, egg white F1, egg yolk F75, milk F2, cod F3, wheat F4, crab F23, shrimp F24, beef F27, mutton F28 and cross-reactive carbohydrate determinant CCD. This embodiment can simultaneously detect 24 specific IgE antibodies in the same reaction tube.

[0087] Example 10

[0088] In the detection reagent of the allergen-specific IgE antibody quantitative detection kit, the coded microsphere mixture includes a second group of comprehensive antigens, and the second group of comprehensive antigens includes a first group of inhaled antigens, a second group of inhaled antigens, a first group of food antigens and a second group of food antigens, specifically including house dust mite D1, Der p 1, Der p 2, Der p 10, Der p 23, dust mite D2, cockroach I6, Aspergillus fumigatus M3, Alternaria alternata M6, cat dander E1, Fel d 1, dog dander E5, Can f 1, common ragweed W1, Artemisia argyi W6, Art v 1, Bermuda grass G2, Timothy grass G6, white birch T3, maple sycamore T11, willow T12, egg white F1, egg yolk F75, Gal d 1, Gal d 2, Gal d 3, Gal d4, milk F2, Bos d 4, Bos d 5, Bos d 6, Bos d 8, cod F3, wheat F4, crab F23, shrimp F24, beef F27, mutton F28 and cross-reactive carbohydrate determinant CCD. This embodiment can simultaneously detect 39 specific IgE antibodies in the same reaction tube.

[0089] 3. Preparation method of liquid chip detection reagents, detection process and results

[0090] The embodiment of the present invention provides an allergen-specific IgE antibody detection kit based on a liquid phase chip method, the kit includes a coded microsphere mixture, a fluorescent conjugate, a biotin marker, a reaction buffer, a washing buffer and a detection solution, a calibrator and a quality control product, and can simultaneously detect multiple allergen-specific IgE antibodies. Table 1 shows the main components of the allergen-specific IgE antibody detection kit of the present invention. The kit is for 50 and 100 people, and the universal solutions such as the washing buffer and the detection solution are phosphate buffer containing Tween20.

[0091] Table 1. The main components of the allergen-specific IgE antibody detection kit of the present invention.

[0092]

[0093] The coded microsphere mixture of the detection reagent is a mixture of allergen-coated coded microspheres (Examples 1 to 10) and quality control microspheres, wherein the quality control microspheres include positive quality control microspheres and negative quality control microspheres, wherein the positive quality control microspheres are DNP-BSA-coated coded microspheres, and the negative quality control microspheres do not contain coatings.

[0094] The coding microsphere mixed solution of the calibration curve reagent is a mixed solution of mouse anti-human IgE monoclonal antibody coated coding microspheres and quality control microspheres, and the quality control microspheres include positive quality control microspheres and negative quality control microspheres.

[0095] The fluorescent conjugate is allophycocyanin (APC) with an emission wavelength of 620nm to 760nm or streptavidin labeled with Alexa Fluor 647 or Cy5, preferably streptavidin labeled with allophycocyanin;

[0096] Taking allophycocyanin as an example, the preparation method of the fluorescent conjugate includes: coupling streptavidin with allophycocyanin to obtain a fluorescent conjugate, and preparing the streptavidin labeled with allophycocyanin with a 0.1M phosphate buffer (pH=7-8) containing 0.5-3% human serum albumin, 1-10% trehalose, 0.05-0.2% Tween-20, and 0.06-0.3% Proclin 300 to prepare each index to 0.1-5.0 μg / mL, thereby obtaining the streptavidin labeled with allophycocyanin;

[0097] The biotin-labeled anti-human IgE antibody is a biotin-labeled mouse anti-human IgE antibody, and the biotin-labeled anti-DNP antibody is a biotin-labeled mouse anti-DNP monoclonal antibody. The preparation method is to take an appropriate amount of anti-human IgE antibody or anti-DNP antibody, add NHS-activated biotin, incubate at room temperature for 0.5 to 2 hours, dialyze with 20mM PBST as the dialysate, and store in a phosphate buffer containing human serum albumin. The concentration of each indicator of the biotin-labeled anti-human IgE antibody is 0.1 to 5.0 μg / mL, and the concentration of the biotin-labeled anti-DNP antibody is 0.1 to 5.0 μg / mL;

[0098] The reaction buffer and the detection solution contain a phosphate buffer containing Tween-20 and Procline 300;

[0099] The calibrator is IgE protein, and the concentrations of the calibrator include 0AU / mL, 0.1AU / mL, 0.35AU / mL, 0.70AU / mL, 3.5AU / mL, 17.5AU / mL, 50AU / mL, and 100AU / mL.

[0100] The quality control products are specific IgE curve quality control products and specific IgE quality control products. The specific IgE curve quality control products are IgE proteins. The specific IgE quality control products realize the indoor quality control of allergen-specific IgE through three IgE antibody sera containing high, medium and low concentrations, such as specific IgE multiple negatives, with concentration ranges of 0.35-3.5AU / mL, 3.5-17.5AU / mL, and 17.5-100AU / mL for house dust mite (D1) IgE antibodies, birch (T3) IgE antibodies, and milk (F2) IgE antibodies. Or the specific IgE negative serum is diluted to the house dust mite (D1) IgE antibodies, birch (T3) IgE antibodies, and milk (F2) IgE antibodies IgE antibody sera in the above three concentration ranges, which are quality control products.

[0101] The curve quality control product is IgE protein, and the concentrations of the curve quality control product include 0.7AU / mL and 17.5AU / mL.

[0102] The present invention provides a detection method using the above-mentioned allergen-specific IgE antibody quantitative detection kit. Preferably, the detection method uses the iMatrix fully automatic liquid chip analyzer of Zhonghan Shengtai Biotechnology Co., Ltd. to perform fully automated detection; comprising the following detection steps:

[0103] S1. Add the sample to be tested (or calibrator and quality control product), reaction buffer, and the coded microsphere mixture and anti-human IgE monoclonal antibody coated coded microsphere working solution to the reaction tube, stir and mix evenly, and react at 37°C;

[0104] S2. After the reaction is completed, magnetic separation is performed, the supernatant is discarded, and a washing buffer is added. After stirring and mixing, magnetic separation is performed again, and the supernatant is discarded again; the washing step is repeated three times in total;

[0105] S3, then add biotin marker to the reaction tube, stir and mix well, and then react at 37°C;

[0106] S4, repeat the cleaning steps of S2 three times in total;

[0107] S5, then add the fluorescent conjugate to the reaction tube, stir and mix well, and then react at 37°C;

[0108] S6, repeat the cleaning step of S2 three times in total;

[0109] S7, adding an equal volume of the detection solution to the reaction tube, stirring to mix, and placing the reaction tube in a flow cytometry luminometer for reading to determine the fluorescence value;

[0110] S8. Use the calibrator to obtain a calibration curve, and the iMatrix analysis software will enter the fluorescence value into the formula to calculate the concentration value of the allergen-specific IgE antibody in the sample to be tested.

[0111] S9, S1~S8 use iMatrix fully automatic liquid chip analysis instrument to achieve automatic detection. It can also be operated manually and combined with conventional flow cytometry detection.

[0112] In the above-mentioned Example 9, 1 AU / mL=1 IU / mL, which can be traced back to the third generation international standard 11 / 234 of human serum immunoglobulin E of the World Health Organization (WHO).

[0113] The embodiments 9 and 10 were used to evaluate the analytical performance of the reagents.

[0114] Evaluation of the level consistency rate and negative and positive compliance rates in Example 1.

[0115] In the embodiment 10, more than 40 samples were selected for each project, and all samples were tested using Phadia or Haike biological detection kit as comparison reagents, with 0.35AU / mL as the reference value. The evaluation results are shown in Table 2. The ±1 level compliance rate is basically greater than 90%, and the total compliance rate is basically greater than 85%.

[0116] Table 2 Summary of consistency and compliance rates

[0117]

[0118] Effect Example 2 Minimum Detection Limit Evaluation

[0119] The negative samples were tested 20 times in parallel, and the average value plus 2 times the standard deviation was taken for the test results. In Example 10, the minimum detection limit obtained was no more than 0.2AU / mL. In Example 9, the minimum detection limit obtained was no more than 0.1AU / mL, reaching the sensitivity of fluorescent enzyme-linked immunosorbent assay or chemiluminescence assay.

[0120] Effect Example 3 Linear Range

[0121] Example 10, detection was performed at 10 concentration points within the concentration range of 0.1-100 AU / mL, and the linear correlation coefficient r≥0.9900.

[0122] Effect Example 4 Precision Evaluation

[0123] In Example 10, the precision reference material was tested 10 times in parallel, and the coefficient of variation (CV) of the test results was calculated to be ≤10%.

[0124] Effect Example 5 Specificity Evaluation

[0125] Example 10: Hemoglobin <400 mg / L, triglyceride or cholesterol <1500 mg / dL, bilirubin <5.0 mg / dL, rheumatoid factor <1000 IU / mL in the sample have no effect on the test results. IgA solution, IgM solution and IgG solution (content not less than 400 μg / mL) prepared from negative samples were tested, and the results were all no higher than 0.2 AU / mL; samples containing a total IgE concentration of not less than 100 IU / mL and no specific antibodies were tested, and the results were all no higher than 0.2 AU / mL.

[0126] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A quantitative detection kit for allergen-specific IgE antibodies based on a liquid phase chip method, characterized in that: include: Capture probe-coated coded microspheres, fluorescent conjugates, biotin markers, reaction buffers, calibrators and quality control products; wherein, The capture probe includes an antigen or an anti-human IgE monoclonal antibody, wherein the antigen includes an allergen and an allergen component antigen, and the allergen and the allergen component antigen are coated on coding microspheres to form allergen-coated coding microspheres and allergen component antigen-coated coding microspheres respectively; the anti-human IgE monoclonal antibody is coated on magnetic coding microspheres to form anti-human IgE monoclonal antibody-coated coding microspheres, which are used for specific IgE calibration curves; The coded microsphere is an assembled microsphere structure including a mother ball and daughter balls located on the surface of the mother ball.

2. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The allergens are extracted from house dust mites, dust mites, cockroaches, Aspergillus fumigatus, Alternaria alternata, cat dander, dog dander, common ragweed, mugwort, timothy grass, Bermuda grass, birch, maple sycamore, willow, egg white, egg yolk, milk, cod, wheat, crab, shrimp, beef, lamb and cross-reactive carbohydrate determinants; The allergen component antigens are highly purified naturally extracted antigens or genetically engineered recombinant antigens, and are from Der p 1, Der p 2, Der p 10, Der p 23, Fel d 1, Can f 1, Art v 1, Gal d 1, Gal d 2, Gal d 3, Gald4, Bos d 4, Bos d 5, Bos d 6, and Bos d 8.

3. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The allergen is an allergen modified by a hydrophilic polymer, the hydrophilic polymer combines with the allergen to form a complex, the hydrophilic polymer is poly-L-lysine, nona-arginine, polyethyleneimine, preferably poly-L-lysine, and its molecular weight is 1000-20000Da; the molar ratio of the hydrophilic polymer to the allergen is 1:50-1:300, preferably 1:180-1:

210.

4. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The coded microspheres are carboxyl coded microspheres or toluenesulfonyl coded microspheres, preferably carboxyl coded microspheres.

5. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: It also has a quality control coded microsphere system, which includes multiple coded microspheres coated with quality control products. The quality control coded microspheres include at least one of internal quality control coded microspheres and blank control quality control coded microspheres. The internal quality control coded microspheres (i.e., positive quality control microspheres) are used to detect the instrument detection system and reagent conditions, and the blank control quality control coded microspheres (i.e., negative quality control microspheres) are used to detect non-specific binding of samples. The quality control coded microsphere system and the detection coded microsphere system (a mixture of coded microspheres coated with different capture probes) are in the same reaction tube and are independent of each other.

6. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The fluorescent conjugate is streptavidin labeled with a fluorescent material having an emission wavelength of 620nm to 760nm; the fluorescent material is selected from allophycocyanin, Alexa Fluor 647 or Cy5.

7. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The biotin marker includes biotin-labeled anti-human IgE antibody and anti-DNP antibody. The biotin-labeled anti-human IgE antibody is a biotin-labeled mouse anti-human IgE antibody, and the biotin-labeled anti-DNP antibody is a biotin-labeled mouse anti-DNP monoclonal antibody.

8. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The reaction buffer is a phosphate buffer containing Tween-20 and Procline300.

9. The allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method according to claim 1, characterized in that: The calibrator is IgE protein, and the concentrations of the calibrator include 0AU / mL, 0.1AU / mL, 0.35AU / mL, 0.70AU / mL, 3.5AU / mL, 17.5AU / mL, 50AU / mL, and 100AU / mL; The quality control products are specific IgE curve quality control products and specific IgE quality control products. The specific IgE curve quality control product is IgE protein. The specific IgE quality control product realizes the indoor quality control of allergen-specific IgE through three IgE antibody sera containing high, medium and low concentrations.

10. Use of the allergen-specific IgE antibody quantitative detection kit based on the liquid phase chip method as claimed in claim 1 in the preparation of products for allergy diagnosis and prognosis.

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